All the clinical and pathologic features of malaria are attributed solely to the parasitic stages of the asexual erythrocytic cycle which occurs in the vertebrate host. The propagation of this cycle in a host is dependent upon extracellular merozoites attaching to and invading susceptible erythrocytes (red blood cells). This attachment and initiation of invasion by malaria merozoites is mediated through specific interactions between parasite receptors and ligand molecules on the erythrocyte plasma membrane. Butcher, G. A., et al, Nature 244:40, 1973; Miller, L. B., et al, J. Exp. Med. 138: 1597, 1983.
The invasion of host erythrocytes by parasite merozoites involves a defined series of events (Bannister, L. H., et al., Parasitology 71:483-491, 1975; Dvorak, J. A., et al., Science, 187:748-749, 1975; Aikawa, et al., J. Cell Biol., 77:72-82, 1978) which include: 1) initial recognition and attachment of the merozoite to the erythrocyte membrane; 2) orientation of the merozoite so that its apical end is apposed to (i.e. oriented towards) the erythrocyte membrane; 3) formation of a junction between the apical end of the merozoite and the erythrocyte; 4) invagination of the erythrocyte membrane to form a vacuole; 5) movement of the merozoite into the vacuole by a moving Junction around the merozoite; 6) closure of the erythrocyte and vacuole membranes resulting in the parasite residing within a parasite-bearing vacuole in the host erythrocyte. This process must involve the complex interaction of numerous components of both parasite and host origin.
In principle, strategic interference with one or more of the foregoing invasion events could prevent invasion, thereby precluding the intra-erythrocytic propagation of the parasite, and eventually diminishing or abolishing infection. However, despite considerable research efforts worldwide, the complexity of the parasite and of its relationship with its host, it has not yet been possible to discover a satisfactory solution for prevention or abatement of the blood stage of malaria. Accordingly, there is a felt need in the field for materials and methods that could be used to accomplish such a goal (e.g. by directly interfering with invasion or by elucidating the mechanism of erythrocyte invasion and assisting in the identification of means for raising preventive and therapeutic barriers against such invasion).
The malarial species P. vivax, one of the four species infective to humans, is a particularly difficult target for such efforts. This parasite is in, short supply and cannot be cultured in vitro, as has been possible with P. knowlesi (a simian malaria parasite) and P. falciparum (another human malaria parasite). Although P. vivax bears substantial phylogenetic similarity to P. knowlesi, the two species are different in many important respects. For example, P. vivax is not infective at all to many simian species and infection is poorly established in others, whereas P. knowlesi is poorly infective to humans while readily infecting many simian species.
The preinvasion orientation of malarial merozoites (such that the epical end is apposite to the erythrocyte surface) indicates that the epical end plays an important role in the invasion process but this role is complex and is not yet clearly understood. Therefore, identification of proteins or other structural features specific to the epical end will provide a better understanding of the molecular mechanism of the invasion process.
Moreover, antigens specifically associated with the apical end are likely to play an important role in apical-end functions, including but not limited to the invasion process and particularly its substages (3) and (4) mentioned above, and therefore at least some such antigens may constitute targets for new antimalarial drugs and/or potential candidates for new vaccines against malaria.
Antibodies raised against an epical end-associated antigen would be useful in studies of parasite morphology and structure; could be used in diagnostic or other serological assays designed to determine infection and measure its extent; and could be also used to inhibit invasion by interfering with erythrocyte binding of the apical antigen or otherwise impeding the function of an apical antigen indispensable to the invasion process.
Human beings lacking the Duffy blood group (Marsh, W. L., CRC Crit. Rev. Clin. Lab. Sci. 5:387-412, 1975) on the surface of their erythrocytes are refractory to infection by Plasmodium vivax (Miller, L. H., et al, N. Engl. J. Med. 295: 302-304, 1976). These erythrocytes are not susceptible to invasion by either P. vivax (Barnwell, J. W., et al., J. Exp. Med. 169:1795, 1989 or the phylogenetically related, and more easily maintained, simian malaria, P. knowlesi (Miller, L. H., et al., Science 189:561-563, 1975; Mason, S. J., et al., Br. J. Haematol. 36:327-335, 1977), in vitro. P. knowlesi merozoites will attach to and orient their apical ends towards the Duffy-negative erythrocyte membrane but no junction is formed between the cells (Miller, L. H., et al., J. Exp. Med. 149: 172-185, 1979. These observations lead to the hypothesis that the 35-46 kilodalton (kD) Duffy blood group glycoprotein (Hadley, T. J., et al., Science 223:597-599, 1984) is an essential ligand in the invasion process of human erythrocytes by P. vivax. (A P. falciparum merozoite antigen has been identified but it binds to glycophorin.) This hypothesis was further supported by the ability of polyclonal antisera against Fy.sup.a Duffy determinant and proteolytic cleavage of the Duffy determinants from erythrocytes to inhibit P. knowlesi invasion of human erythrocytes (Miller, L. H., et al.; Mason, S. J., et al., supra). The specificity of this interaction has been demonstrated by blocking the ability of P. vivax merozoites to invade human erythrocytes with both intact and F(ab) fragments of an anti-Duffy monoclonal antibody (Barnwell, J. W., et al., supra) directed against the newly described Fy.sup.6 determinant of the Duffy glycoprotein (Nichols, M. E., et al., J. Exp. Med. 166:776-785, 1987).
Accordingly, the present inventors sought to take advantage of this interaction between P. vivax or P. knowlesi merozoites and the erythrocyte surface (including, but not limited to, the Duffy glycoprotein) to elucidate the molecular nature of Plasmodium invasion of host erythrocytes and to devise means and methods useful against malaria.